7. "Staining"
7.1. Introductory Remarks
When one speaks of "staining" for electron microscopy, he is thinking
of the addition of heavy atoms to the specimen. These scatter electrons
more than light atoms, and so enhance the final contrast of the picture.
When osmium tetroxide is the fixative, it is fair to say that this substance
also acts as a stain in proportion to the bound osmium. Indeed, electron
microscopists, more or less seriously, have complained that the micrographs we finally study represent little more than patterns of osmium
(or other heavy metal) deposition. One has only to observe and consider
the low contrast of unstained material fixed with aldehyde to realize
that there is a large measure of truth in such statements.
So long as methacrylate embedments were used nearly exclusively for
ultrathin sectioning osmium tetroxide served quite effectively as a general
purpose stain, for it does combine with most cytological components
in one way or another. However, as has already been stated (Chapter 4.9),
methacrylate decomposes to a considerable extent during electron bombardment, so that the contrast differential between the specimen and
the plastic background becomes enhanced. This does not happen when
epoxy or polyester plastics are used (Chapter 4.9), and then it is empirically obvious that insufficient amounts of osmium ordinarily are
bound to tissue components to serve effectively as a stain. Indeed it is
fair to say that the recent shift to these thermally stable, cross-linked
plastics to achieve better preservation of fine structure depended upon
the almost simultaneous development of adequate stains to use with
them. Without the new stains the spectacular progress of the last five
years would have been impossible.
During the "methacrylate era" phosphotungstic acid was the only
commonly used additive stain. This had been introduced by Hall et al.
(1945) in their studies of collagen. Wohlfarth-Bottermann (1956) employed it as a tissue stain. Of course sporadic efforts were made to use
other compounds containing heavy atoms, but the results rarely were
convincing. Then Watson (1958) made a comparative study of a number
of heavy atom compounds that hopefully might be attached to cyto216
7.1. Introductory Remarks
When one speaks of "staining" for electron microscopy, he is thinking
of the addition of heavy atoms to the specimen. These scatter electrons
more than light atoms, and so enhance the final contrast of the picture.
When osmium tetroxide is the fixative, it is fair to say that this substance
also acts as a stain in proportion to the bound osmium. Indeed, electron
microscopists, more or less seriously, have complained that the micrographs we finally study represent little more than patterns of osmium
(or other heavy metal) deposition. One has only to observe and consider
the low contrast of unstained material fixed with aldehyde to realize
that there is a large measure of truth in such statements.
So long as methacrylate embedments were used nearly exclusively for
ultrathin sectioning osmium tetroxide served quite effectively as a general
purpose stain, for it does combine with most cytological components
in one way or another. However, as has already been stated (Chapter 4.9),
methacrylate decomposes to a considerable extent during electron bombardment, so that the contrast differential between the specimen and
the plastic background becomes enhanced. This does not happen when
epoxy or polyester plastics are used (Chapter 4.9), and then it is empirically obvious that insufficient amounts of osmium ordinarily are
bound to tissue components to serve effectively as a stain. Indeed it is
fair to say that the recent shift to these thermally stable, cross-linked
plastics to achieve better preservation of fine structure depended upon
the almost simultaneous development of adequate stains to use with
them. Without the new stains the spectacular progress of the last five
years would have been impossible.
During the "methacrylate era" phosphotungstic acid was the only
commonly used additive stain. This had been introduced by Hall et al.
(1945) in their studies of collagen. Wohlfarth-Bottermann (1956) employed it as a tissue stain. Of course sporadic efforts were made to use
other compounds containing heavy atoms, but the results rarely were
convincing. Then Watson (1958) made a comparative study of a number
of heavy atom compounds that hopefully might be attached to cyto216